Antimicrobial Susceptibility Patterns of Ureaplasma urealyticum and Mycoplasma hominis Isolated From Pregnant Women.

BACKGROUND: Mycoplasma hominis and Ureaplasma urealyticum bring with them an increased risk of pregnancy complications, such as premature membrane rupture, vaginitis and preterm birth.
OBJECTIVES: The present investigation was carried out to study the prevalence of M. hominis and U. urealyticum in pregnant women and to study their resistance against commonly used antibiotics.
MATERIALS AND METHODS: Three hundred and fifty high vaginal swabs were taken from pregnant women. Commercial Mycoplasma IST-2 kit was used for bacterial isolation. The results of the kits were confirmed using the PCR. The pattern of antibiotic resistance was determined using the disk diffusion method.
RESULTS: Of 350 samples collected, 32 samples (9.14%) were positive for U. urealyticum and 10 samples (2.85%) were positive for M. hominis (P = 0.025). Both U. urealyticum and M. hominis were simultaneously detected in 1.14% of samples. In addition, 40 - 45-year-old pregnant women had the highest levels of U. urealyticum (27.5%), M. hominis (12.5%), and both bacteria (7.5%). U. urealyticum and M. hominis isolates harbored the highest levels of resistance against ciprofloxacin, ofloxacin, erythromycin, and tetracycline. Both isolates were susceptible to pefloxacin, clarithromycin, josamycin, and pristinamycin.
CONCLUSIONS: According to the direct correlation between the increase in the prevalence rate of genital mycoplasmas and increased age of pregnancy, initially, it is better to prevent pregnancy at older ages, and then, should a pregnancy occur, the highest levels of health cares should be provided to older pregnant women.

1. Background

During pregnancy, the body’s immunity levels are reduced. The reduction in the levels of immunity is an important risk factor for the entrance of infectious agents into the vagina. Ureaplasma urealyticum and Mycoplasma hominis, also known as genital mycoplasmas, are commensals that can be detected in the lower genitourinary tract of sexually active women, resulting in colonization of the genitalia by sexual contact (1, 2). Genital mycoplasmas are found in the vaginal milieu of up to 80% of pregnant and non-pregnant women (3). Vaginal colonization of these two pathogenic bacteria mainly cause vaginosis, postpartum fever, pelvic inflammatory disease, infertility, postpartum septicemia, preterm labor, premature rupture of the membranes, systemic neonatal infections, and preterm birth (1-5).

Treatment of diseases caused by U. urealyticum and M. hominis requires antibiotic therapy; however, genital mycoplasmas exhibit an inherent resistance to beta-lactams, glycopeptides, macrolides (erythromycin and azithromycin), lincosamides (clindamycin), and tetracycline antimicrobial agents (6, 7). Tetracycline and quinolones are the drugs of choice, but obstetricians empirically use macrolides for the treatment of pregnant women in many cases (8). However, the pattern of antibiotic resistance in genital mycoplasmas has changed over the time (9). Therefore, it is imperative to recognize the antimicrobial susceptibilities of genital mycoplasmas in each region, and even each hospital, in order to ensure their successful treatment.

2. Objectives

According to the uncertain epidemiology and prevalence of U. urealyticum and M. hominis in Iran, the present investigation was conducted in order to study the prevalence and antimicrobial resistant properties of U. urealyticum and M. hominis isolated from pregnant women who were admitted to the Iranian Obstetrics and Gynecology Health Care Units.

3. Materials and Methods

3.1. Ethical Issues

The ethical committee of various obstetrics and gynecology research centers in hospitals in Iran approved the present study. The authors tried to protect the health, life, integrity, dignity, rights to self-determination, privacy, and confidentiality of all personal information of the studied women. We conform to generally accepted scientific principles, which are based on a thorough knowledge of the scientific literature, other relevant sources of information, and adequate laboratory. All samples were taken from volunteer women who were referred to the obstetrics and gynecology research centers of hospitals in Iran.

3.2. Sample Collection

Between March 2015 and October 2015; 350 high vaginal swab (Copan Diagnostics, Inc, Italy) specimens were taken from pregnant women (range, 22 - 45 years) who referred to the obstetrics and gynecology research centers of the educational hospitals of Iran. Vaginal specimens were collected from the ventral fornix, without any contact with urine or external parts of the reproductive system using a speculum and commercial sterile cotton-tipped swabs. All specimens were collected by an expert midwife.

3.3. Ureaplasma urealyticum and Mycoplasma hominis Isolation

In order to identify the bacteria, the method described by Bayraktar et al. was used. M. hominis and U. urealyticum were detected using a commercial kit, Mycoplasma IST-2 (BioMerieux, Marcy l’Etoile, France), according to the manufacturer’s instructions. (10) The kit contains strips that give information on the presence or absence of M. hominis and U. urealyticum. One strip was placed directly into R1 tubes (transport medium) and subsequently delivered to the clinical laboratory for the identification of both U. urealyticum and M. hominis.

Swabs in the R1 transport medium were processed according to the manufacturer’s instructions. They were vortexed rapidly, and 3 mL of R1 was used to rehydrate the lyophilized growth medium R2 (provided in the Mycoplasma IST-2 kit). A Mycoplasma IST strip, consisting of 22 wells, was then inoculated with the rehydrated R2 growth medium (55 mL per well, overlaid with two drops of mineral oil). From the R2 positive tube, 0.1 mL was also inoculated onto A7 Mycoplasma agar plates (BioMerieux, Marcy l’Etoile, France) and incubated at 37°C in an atmosphere of 5% CO2 to check for characteristic colony morphology. All media and the inoculated strip were incubated at 37°C in a CO2 incubator and observed for color changes, and the results were interpreted after 24 and 48 hours of incubation. Wells provided information on the presence or absence of M. hominis and U. urealyticum, with an estimate of the density of each organism (≥ 104 CFU). The A7 plates were examined with a microscope twice daily for up to five days for characteristic colonies. Colonies presenting with a fried egg appearance suggest the presence of M. hominis, whereas colonies that are brown and tiny indicate the presence of U. urealyticum. M. hominis ATCC 23114 and U. urealyticum ATCC 27618 strains were used as controls.

3.4. PCR Confirmation of Ureaplasma urealyticum and Mycoplasma hominis

A PCR technique was used in order to detect U. urealyticum and M. hominis (11, 12). Total genomic DNA was extracted from the bacterial colonies using a commercial genomic DNA extraction kit (Fermentas, Germany) according to its manufacturer’s instruction. The DNA concentration was determined by measuring the absorbance of the sample at 260 nm using a spectrophotometer (13). Table 1 shows the oligonucleotide primers, the size of products, and PCR conditions used for the detection of U. urealyticum and M. hominis. The PCR amplification products (15 μL) were subjected to electrophoresis in a 1.5% agarose gel in 1X TBE buffer at 80 V for 30 minutes, stained with SYBR Green. All runs included a negative DNA control consisting of PCR grade water, and strains of M. hominis ATCC 23114 and U. urealyticum ATCC 27618 were used as positive controls.

Table 1.

The Oligonucleotide Primers and the PCR Programs Used for Amplification of the Urease Gene of the U. urealyticum and 16SrRNA Gene of the M. hominis

Target Gene/Primer Sequence (5' - 3')	PCR Product, bp	PCR Programs	PCR Volume (50 µL)
Urease gene of the U. urealyticum	429	1 cycle: 95°C; 3 min, 30 cycle: 95°C; 20 s, 58°C; 40 s,72°C; 30 s,1 cycle: 72°C; 8 min	5 µL PCR buffer 10X; 1.5 mM Mgcl2; 200 µM dNTP (Fermentas); 0.5 µM of each primers F and R; 1.25 U Taq DNA polymerase (Fermentas); 2.5 µL DNA template
F-ACGACGT CCATAAGCAACT
R-CAATCTGCTCGTGAAGTATTAC
16S rRNA gene of the M. hominis	344	1 cycle: 95°C; 3 min, 30 cycle: 95°C; 20 s,58°C; 40 s,72°C; 30 s,1 cycle: 72°C; 8 min	5 µL PCR buffer 10X; 1.5 mM Mgcl2; 200 µM dNTP (Fermentas); 0.5 µM of each primers F and R; 1.25 U Taq DNA polymerase (Fermentas); 2.5 µL DNA template
F- CAA TGG CTA ATG CCG GAT ACG C
R-GGT ACC GTC AGT CTG CAA T

3.5. Antimicrobial Susceptibility Testing

Patterns of antimicrobial resistance were studied using the simple disk diffusion technique. The Mueller–Hinton agar (Merck, Germany) medium was used for this purpose. Antibiotic resistance of M. hominis and U. urealyticum strains against 11 commonly used antibiotics, including tetracycline (30 µg/disk), clindamycin (2 µg/disk), doxycycline (30 µg/disk), pefloxacin (5 µg/disk), ofloxacin (5 µg/disk), erythromycin (15 µg/disk), clarithromycin (2 µg/disk), azithromycin (15 µg/disk), Josamycin (30 µg/disk), ciprofloxacin (5 µg/disk), and pristinamycin (15 µg/disk) antibiotic agents (Oxoid, UK) were analyzed using the Clinical Laboratory Standard Institute protocol (CLSI) (14). M. hominis ATCC 23114 and U. urealyticum ATCC 27618 were also used as positive controls.

3.6. Statistical Analysis

Statistical analysis was performed using SPSS/16.0 software for significant relationships. The incidences of M. hominis and U. urealyticum and the prevalence of antibiotic resistance in the pregnant women of various ages were statistically analyzed. Statistical significance was regarded at a P value < 0.05.

4. Results

The results of the present study showed the high prevalence of U. urealyticum and M. hominis in the high vaginal swab samples of pregnant women. Table 2 represents the total prevalence of U. urealyticum and M. hominis in the high vaginal swab samples of pregnant women. Of 350 samples collected for this study, 32 samples (9.14%) were positive for U. urealyticum, and 10 samples (2.85%) were positive for M. hominis. Both U. urealyticum and M. hominis were recovered from 4 high vaginal swab samples (1.14%). The results of the conventional technique were also confirmed by the PCR method (Figures 1 and 2). Statistically significant differences were seen between the prevalence of U. urealyticum and M. hominis (P = 0.025). Our results revealed that 40 - 45-year-old pregnant women had the highest levels of U. urealyticum (27.5%), M. hominis (12.5%), and both bacteria (7.5%). Statistically significant differences were seen for the prevalence of U. urealyticum between 40 - 45 year olds, 20 - 25-year-old pregnant women (P = 0.032), and between 35 - 40 year olds and 20 - 25-year-old pregnant women (P = 0.039). Statistically significant differences were seen for the prevalence of M. hominis between 40 - 45-year-old and 20 - 25-year-old pregnant women (P = 0.044).

Table 2.

Total Distribution of U. urealyticum and M. hominis in the High Vaginal Swab Samples of Pregnant Women

Samples/Age Groups, y	No. Samples Collected	No. Pathogenic Bacteria, %
		U. urealyticum	M. hominis	Both Bacteria
High vaginal swabs
20 - 25	100	3 (3)
25 - 30	100	4 (4)	1 (1)
30 - 35	60	6 (10)	1 (1.66)
35 - 40	50	8 (16)	3 (6)	1 (2)
40 - 45	40	11 (27.5)	5 (12.5)	3 (7.5)
Total	350	32 (9.14)	10 (2.85)	4 (1.14)

Figure 1.

PCR Gel Electrophoresis for the Detection of the Urease Gene of the U. urealyticum in the High Vaginal Swab Samples of Pregnant Women

M, 100 bp ladder; 1, Positive sample for urease gene of the U. urealyticum (422 bp); 2, Positive control; 3, Negative control.

Figure 2.

PCR Gel Electrophoresis for Detection of 16S rRNA Gene of the M. hominis in the High Vaginal Swab Samples of Pregnant Women

M, 100 bp ladder; 1, Positive sample for 16S rRNA gene of the M. hominis (344 bp); 2, Positive control; 3, Negative control.

Table 3 shows the prevalence of antibiotic resistance in U. urealyticum and M. hominis isolated from the high vaginal swab samples of pregnant women. U. urealyticum isolates of our study harbored the highest levels of resistance against ciprofloxacin (78.12%), ofloxacin (62.5%), erythromycin (56.25%), and tetracycline (50%). M. hominis showed similar resistance pattern with different percentages. M. hominis isolates in our study harbored the highest levels of resistance against ciprofloxacin (70%), ofloxacin (60%), erythromycin (40%), and tetracycline (40%). Statistically significant differences were seen for the prevalence of U. urealyticum resistance between tetracycline and pristinamycin (P = 0.026), ofloxacin and pristinamycin (P = 0.038), and ciprofloxacin and Josamycin (P = 0.043). Statistically significant differences were seen for the prevalence of M. hominis resistance between ciprofloxacin and pristinamycin (P = 0.035) and ofloxacin and Josamycin (P = 0.040).

Table 3.

Antibiotic Resistance Patterns of U. urealyticum and M. hominis Isolated From the High Vaginal Swab Samples of Pregnant Women[a]

Type of Bacteria (No. Positive Isolates)	Pattern of Antibiotic Resistance, %
	Tet30	Cln2	Dox30	Pfl5	Ofl5	Ert15	Clr2	Azt15	Jsm30	Cip5	Prst15
U. urealyticum (32)	16 (50)	10 (31.25)	7 (21.87)	11 (34.37)	20 (62.5)	18 (56.25)	9 (28.12)	13 (40.62)	5 (15.62)	25 (78.12)	4 (12.5)
M. hominis (10)	4 (40)	3 (30)	2 (20)	2 (20)	6 (60)	4 (40)	2 (20)	3 (30)		7 (70)

aAbbreviations: Azt15, azithromycin (15 µg/disk); Cip5, ciprofloxacin (5 µg/disk); Cln2, clindamycin (2 µg/disk); Clr2, clarithromycin (2 µg/disk); Dox30, doxycycline (30 µg/disk); Ert15, erythromycin (15 µg/disk); Jsm30, Josamycin (30 µg/disk); Ofl5, ofloxacin (5 µg/disk); Pfl5, pefloxacin (5 µg/disk); Prst15, pristinamycin (15 µg/disk) antibiotic agents (Oxoid, UK); Tet30, tetracycline (30 µg/disk).

5. Discussion

The results of the present study showed the important public health issue regarding the high prevalence of resistant strains of U. urealyticum and M. hominis in Iranian pregnant women. Higher prevalence of U. urealyticum; higher prevalence of both bacteria in 40 - 45-year-old women; and the high prevalence of resistance against ciprofloxacin, ofloxacin, erythromycin, and tetracycline were imperative findings in this study. In total, four women (1.14%) were positive for both genital mycoplasmas. In contrast, no 20 - 25-year-old women harbored M. hominis, and there were no positive results for both genital mycoplasmas together in the 20 - 25, 25 - 30, or 30 - 35-year-old cohorts.

Our results showed a lower prevalence rate of genital mycoplasmas when compared with those of many previous studies, which had reported the prevalence of U. urealyticum as between 10% and 50% and the prevalence of M. hominis as less than 30% (15-17). Our results showed that U. urealyticum was more commonly detected than M. hominis in pregnant women. Our findings are impartially consistent with those of other studies conducted in Poland (18), Turkey (10), and Greece (19), but definitely different to those of Papua New Guinea (20), Japan (21), and Portugal (15). Simultaneous colonization with both U. urealyticum and M. hominis was not common (1.14% in our study), but has been found to be as low as 2.92% in one population (19). The exact cause for the concurrent isolation of U. urealyticum and M. hominis in the high vaginal swab samples remains unclear, but it may be related to vaginal environment, sexual activity, socioeconomic status, sexual education, altered immune status, and poor hygiene (18, 22). In a study conducted by Diaz et al. genital mycoplasmas were detected in 63.1% of high vaginal samples, and among these, U. urealyticum was detected in 68.9%, M. hominis alone in 4.3%, and both bacteria in 26.7%, which was entirely higher than our results (23). Koh et al. reported that U. urealyticum was detected in 38.6% of samples, M. hominis was detected in 1.8%, and both bacteria were detected in 6.7% of samples taken from pregnant women, which also was entirely higher than our results (24).

Some of the main reasons for the various prevalence of U. urealyticum and M. hominis in the samples taken from the vagina of women in various studies are the fact that perhaps the type of samples, number samples collected, method of sampling, method of experiment, age of women, and geographical distribution were different in each study. A change in vaginal pH (such as bleeding in pregnancy, vaginal douching, or sexual intercourse) may incline to an overgrowth of U. urealyticum and M. hominis (25, 26). This can be another reason for the different prevalences of bacteria in various studies.

The high prevalence of antibiotic resistance in the vaginal isolates of U. urealyticum and M. hominis has been reported in many studies. Increased resistance in genital mycoplasmas is reported with global variations to the choice drugs (doxycycline and tetracycline) (23, 24, 27). Our findings of revealed that at least half of the U. urealyticum and M. hominis were resistant to ciprofloxacin, ofloxacin, erythromycin, and tetracycline. This part of our study was similar to those conducted in Germany (27), Mexico (28), and Greece (19). Diaz et al. reported that less than 35% of U. urealyticum isolates in Cuba were resistant to minocycline, pefloxacin, doxycycline, tetracycline, clindamycin, and azithromycin (23). They showed that resistance against ofloxacin, clarithromycin, and erythromycin were 64.3%, 63%, and 46.1%, respectively, which was entirely similar to our findings. Bayraktar et al. in a study conducted in Turkey on pregnant women, reported that the prevalence of resistance of genital mycoplasmas against doxycycline, josamycin, ofloxacin, erythromycin, tetracycline, ciprofloxacin, azithromycin, clarithromycin, and pristinamycin were 0%, 0%, 81.3%, 34.4%, 0%, 84.4%, 25%, 12.5%, and 0%, respectively, which was the report with the most similarity to our study (10). Redelinghuys et al. in a study conducted on South African pregnant women, reported that susceptibilities of Ureaplasma spp. to levofloxacin and moxifloxacin were 59% and 98%, respectively. They showed that mixed isolates (Ureaplasma species and M. hominis) were highly resistant to erythromycin (97%) and tetracycline (97%) (29). Differences in the ideas of gynecologists about antibiotic prescription causes variations in the levels of antibiotic resistance against different antibiotics. In addition, the differences in the bactericidal activities of antibiotics, and differences in the difficulty of developing resistance against various antibiotics, are two other reasons for differences in the levels of antibiotic resistance. In the other than, excessive and indiscriminate prescription of ciprofloxacin, ofloxacin, erythromycin and tetracycline antibiotics caused to U. urealyticum and M. hominis strains of our research had such high levels of resistance.

5.1. Conclusion

In conclusion, the isolation rate of U. urealyticum and M. hominis in Iranian pregnant women was 9.14% and 2.85%, respectively. Based on the higher prevalence of both bacteria in 40 - 45-year-old pregnant women, and also according to presence of a direct correlation between the increase in the prevalence rate and increase in the age of pregnancy, it is better to prevent pregnancy at older ages. Our results revealed that the highest levels of health care should be provided for older pregnant women. Both isolates were resistant to ciprofloxacin, ofloxacin, erythromycin, and tetracycline, but susceptible to pefloxacin, clarithromycin, josamycin, and pristinamycin. Typically, susceptibility of isolates azithromycin, the empirical treatment regimen for pregnant women in our geographic region, was not as high as we expected. Our results showed that empirical treatment without the isolation and identification of genital U. urealyticum and M. hominis would fail in many cases. Determination of the antimicrobial susceptibility of the U. urealyticum and M. hominis by simple methods like disk diffusion technique is required to avoid therapeutic failures.